Conventionally, the following method has been used for isolating and culturing cells derived from human and animal sources. Firstly, a culture solution is prepared by adding nutrient such as glucose, a growing agent for promoting growth of a cell, antibiotics for preventing proliferation of miscellaneous bacteria, or the like, into a physiological saline solution. Then, cells are scattered in the culture solution at a predetermined concentration. Then, the culture solution containing cells is placed in a container such as a plane culture dish. Thereafter, the culture dish is set in an environment maintaining device (generally referred to as an incubator) capable of maintaining a surrounding environment. With the environment maintaining device, temperatures, carbon dioxide concentrations, and oxygen concentrations as the surrounding environment of the culture dish are kept at predetermined values and allowed to stand for two to three days so as to wait for division and proliferation of the cells.
The proliferation rate of a cell varies depending upon types, states and environments of a cell. In, for example, RBL (Rat Basophilic Leukemia), the cell concentration becomes about 10 times after three days have passed. At this time, the cell absorbs nutrient necessary for proliferation from a culture solution around the cell and, at the same time, the cell exhausts waste matters. Therefore, the state of the culture solution in the culture dish is largely changed as cell proliferation proceeds. Furthermore, in, for example, CHO (Chinese Hamster Ovary), cells proliferate while they adhere to a plane part of the culture dish. Therefore, when a space to which the cells adhere is lost in the plane part of the culture dish, the proliferation of cells stops. In this way, when the proliferation of cells proceeds, the density of cells in the culture solution is increased, and further proliferation is not carried out. Therefore, after the culture of cells proceeds to some extent, the cells and the culture solution are recovered from the culture dish, and the cells are separated from the culture solution by using a centrifugal separator. Then, an old culture solution is removed, and the cells are scattered on a new culture solution at an appropriate concentration. Thus, cells are newly cultured. This operation is generally called subculture.
In particular, it is important for cell culture that the subculture is appropriately carried out according to a state of a cell. However, in particular, in a case of adhesive cells, in a culture dish having a plane part, the lower parts of the cells are closely attached to the plane part. Consequently, it may be difficult to maintain an environment surrounding cells, in particular, an environment surrounding cells on the surface that is brought into contact with the culture dish.
In order to solve such problems, for example, it is proposed that the contacting degree between a culture dish and cells be reduced by forming a group of minute protrusions on a plane part of a culture dish, thus controlling the close attaching degree (for example, PTL 1 and PTL 2).
Alternatively, it is proposed that a plurality of spherical protruding portions, which have been subjected to water-repellent treatment, are arranged on a rectangular plate with an appropriate spacing, and the protruding portions hold a liquid droplet and a cell be cultured in the liquid droplet (for example, PTL 3).
Cells are sensitive to changes of their surrounding environment. Therefore, as mentioned above, failing to exchange culture solutions or maintain a surrounding environment may cause changes in pH or an oxygen concentration of a culture solution, or may cause waste matters to accumulate in a predetermined place. Thus, the activity of cells in the surrounding in which an undesirable change of environment occurs is lost. Therefore, maintaining of the surrounding environment of a cell is an extremely important factor for cell culture. However, in a conventional plane culture dish, the working efficiency mentioned above is not good. Accordingly, in a cell culture substrate having a conventional configuration and a cell culture method using the same, working efficiency of cell culture is not good.
Each of the techniques shown in PTLs 1 to 3 is one of methods for solving such problems, but has limitations on improvement of working efficiency. In the techniques disclosed in PTLs 1 and 2, a group of protrusions are formed by transferring convex and concave patterns on a substrate by pressing a mold provided with minute convex and concave patterns on a substrate as a culture dish. A so-called nano-imprinting technique is used. Therefore, each of the formed group of protrusions is independent from each other. Consequently, a shape in which protrusions are connected to each other cannot be formed. Furthermore, since a group of protrusions formed by the above-mentioned transfer method can be formed only on the plane of the substrate, only a group of protrusions extending in the direction perpendicular to the plane of the substrate can be configured.
Alternatively, in the technique disclosed in PTL 3, similarly, since a rectangular plane substrate is used as a base material, only protrusions extending in the direction perpendicular to the plane of the substrate can be configured. Therefore, formation of protrusions in minute concave and convex patterns formed on the substrate plane of a culture dish has limitations on improvement in the percentage of voids of the protrusions.